scholarly journals Maintenance of Double-Stranded Telomeric Repeats as the Critical Determinant for Cell Viability in Yeast Cells Lacking Ku

2002 ◽  
Vol 22 (7) ◽  
pp. 2182-2193 ◽  
Author(s):  
Serge Gravel ◽  
Raymund J. Wellinger
Keyword(s):  
Dna Damage ◽  
Elevated Temperatures ◽  
Telomeric Repeat ◽  
Telomere Maintenance ◽  
Yeast Cells ◽  
Chromosomal Dna ◽  
End Joining ◽  
Telomeric Repeats ◽  
Critical Determinant ◽  
Dna Damage Checkpoints

ABSTRACT The Saccharomyces cerevisiae Ku complex, while important for nonhomologous DNA end joining, is also necessary for maintaining wild-type telomere length and a normal chromosomal DNA end structure. Yeast cells lacking Ku can grow at 23°C but are unable to do so at elevated temperatures due to an activation of DNA damage checkpoints. To gain insights into the mechanisms affected by temperature in such strains, we isolated and characterized a new allele of the YKU70 gene, yku70-30ts . By several criteria, the Yku70-30p protein is functional at 23°C and nonfunctional at 37°C. The analyses of telomeric repeat maintenance as well as the terminal DNA end structure in strains harboring this allele alone or in strains with a combination of other mutations affecting telomere maintenance show that the altered DNA end structure in yeast cells lacking Ku is not generated in a telomerase-dependent fashion. Moreover, the single-stranded G-rich DNA on such telomeres is not detected by DNA damage checkpoints to arrest cell growth, provided that there are sufficient double-stranded telomeric repeats present. The results also demonstrate that mutations in genes negatively affecting G-strand synthesis (e.g., RIF1) or C-strand synthesis (e.g., the DNA polymerase α gene) allow for the maintenance of longer telomeric repeat tracts in cells lacking Ku. Finally, extending telomeric repeat tracts in such cells at least temporarily suppresses checkpoint activation and growth defects at higher temperatures. Thus, we hypothesize that an aspect of the coordinated synthesis of double-stranded telomeric repeats is sensitive to elevated temperatures.

scholarly journals Recombination Can either Help Maintain Very Short Telomeres or Generate Longer Telomeres in Yeast Cells with Weak Telomerase Activity

2011 ◽  
Vol 10 (8) ◽  
pp. 1131-1142 ◽  
Author(s):  
Evelina Basenko ◽  
Zeki Topcu ◽  
Michael J. McEachern
Keyword(s):  
Homologous Recombination ◽  
Telomere Length ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Yeast Cells ◽  
Colony Morphology ◽  
Wild Type ◽  
Telomeric Repeats ◽  
Yeast Mutants ◽  
Derivatives Of

ABSTRACT Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles.

scholarly journals Requirement for End-Joining and Checkpoint Functions, but NotRAD52-Mediated Recombination, after EcoRI Endonuclease Cleavage of Saccharomyces cerevisiaeDNA

1998 ◽  
Vol 18 (4) ◽  
pp. 1891-1902 ◽  
Author(s):  
L. Kevin Lewis ◽  
Jakob M. Kirchner ◽  
Michael A. Resnick
Keyword(s):  
Nuclear Dna ◽  
Double Strand Breaks ◽  
Yeast Cells ◽  
Chromosomal Dna ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Damaging Agents ◽  
Physical And Chemical ◽  
Type Strains

ABSTRACT RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired inrad52 mutants and in G1-phase Rad+cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly,rad52 mutants were not more sensitive toEcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly whenEcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.

scholarly journals Mycobacterial Nonhomologous End Joining Mediates Mutagenic Repair of Chromosomal Double-Strand DNA Breaks

2007 ◽  
Vol 189 (14) ◽  
pp. 5237-5246 ◽  
Author(s):  
Nicolas C. Stephanou ◽  
Feng Gao ◽  
Paola Bongiorno ◽  
Sabine Ehrt ◽  
Dirk Schnappinger ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Stationary Phase ◽  
Nonhomologous End Joining ◽  
Chromosomal Dna ◽  
Dna Breaks ◽  
Late Stationary Phase ◽  
End Joining ◽  
Dsb Repair ◽  
Break Site

ABSTRACT Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis ΔrecA Δku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.

scholarly journals USP14 is a deubiquitinase for Ku70 and critical determinant of non-homologous end joining repair in autophagy and PTEN-deficient cells

2019 ◽  
Author(s):  
Arishya Sharma ◽  
Turkeya Alswillah ◽  
Isha Kapoor ◽  
Pal Debjani ◽  
Belinda Willard ◽  
...  
Keyword(s):  
Dna Damage ◽  
Degradation Process ◽  
Spectrometric Analysis ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Critical Determinant ◽  
Vitro Assay ◽  
Intracellular Degradation ◽  
Non Homologous End Joining

Abstract Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are predominantly repaired by non-homologous end joining (NHEJ). IR-induced DNA damage activates autophagy, an intracellular degradation process that delivers cytoplasmic components to the lysosome. We identified the deubiquitinase USP14 as a novel autophagy substrate and a regulator of IR-induced DNA damage response (DDR) signaling. Inhibition of autophagy increased levels and DSB recruitment of USP14. USP14 antagonized RNF168-dependent ubiquitin signaling and downstream 53BP1 chromatin recruitment. Here we show that autophagy-deficient cells are defective in NHEJ, as indicated by decreased IR-induced foci (IRIF) formation by pS2056-, pT2609-DNA-PKcs, pS1778-53BP1, RIF1 and a reporter assay activation. Moreover, chromatin recruitment of key NHEJ proteins, including, Ku70, Ku80, DNA-PKcs and XLF was diminished in autophagy-deficient cells. USP14 inhibition rescued the activity of NHEJ-DDR proteins in autophagy-deficient cells. Mass spectrometric analysis identified USP14 interaction with core NHEJ proteins, including Ku70, which was validated by co-immunoprecipitation. An in vitro assay revealed that USP14 targeted Ku70 for deubiquitination. AKT, which mediates Ser432-USP14 phosphorylation, was required for IRIF formation by USP14. Similar to USP14 block, AKT inhibition rescued the activity of NHEJ-DDR proteins in autophagy- and PTEN-deficient cells. These findings reveal a novel negative PTEN/Akt-dependent regulation of NHEJ by USP14.

scholarly journals DNA Damage Checkpoints Inhibit Mitotic Exit by Two Different Mechanisms

2007 ◽  
Vol 27 (14) ◽  
pp. 5067-5078 ◽  
Author(s):  
Fengshan Liang ◽  
Yanchang Wang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Mitotic Exit ◽  
Yeast Cells ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Dna Damage Checkpoints ◽  
Early Anaphase

ABSTRACT Cyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for “Cdc-fourteen early anaphase release”) and mitotic exit network (MEN) pathways. In response to DNA damage, two branches of the DNA damage checkpoint, Chk1 and Rad53, are activated in budding yeast to prevent anaphase entry and mitotic exit, allowing cells more time to repair damaged DNA. Here we present evidence indicating that yeast cells negatively regulate mitotic exit through two distinct pathways in response to DNA damage. Rad53 prevents mitotic exit by inhibiting the MEN pathway, whereas the Chk1 pathway prevents FEAR pathway-dependent Cdc14 release in the presence of DNA damage. In contrast to previous data, the Rad53 pathway negatively regulates MEN independently of Cdc5, a Polo-like kinase essential for mitotic exit. Instead, a defective Rad53 pathway alleviates the inhibition of MEN by Bfa1.

Non-canonical Functions of Telomerase Reverse Transcriptase: Emerging Roles and Biological Relevance

2020 ◽  
Vol 20 (6) ◽  
pp. 498-507 ◽  
Author(s):  
Connor A.H. Thompson ◽  
Judy M.Y. Wong
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Reverse Transcriptase ◽  
Cancer Cells ◽  
Telomere Length ◽  
Telomere Maintenance ◽  
Telomerase Reverse Transcriptase ◽  
Alternative Mechanism ◽  
Dependent Manner ◽  
Mitochondrial Integrity

Increasing evidence from research on telomerase suggests that in addition to its catalytic telomere repeat synthesis activity, telomerase may have other biologically important functions. The canonical roles of telomerase are at the telomere ends where they elongate telomeres and maintain genomic stability and cellular lifespan. The catalytic protein component Telomerase Reverse Transcriptase (TERT) is preferentially expressed at high levels in cancer cells despite the existence of an alternative mechanism for telomere maintenance (alternative lengthening of telomeres or ALT). TERT is also expressed at higher levels than necessary for maintaining functional telomere length, suggesting other possible adaptive functions. Emerging non-canonical roles of TERT include regulation of non-telomeric DNA damage responses, promotion of cell growth and proliferation, acceleration of cell cycle kinetics, and control of mitochondrial integrity following oxidative stress. Non-canonical activities of TERT primarily show cellular protective effects, and nuclear TERT has been shown to protect against cell death following double-stranded DNA damage, independent of its role in telomere length maintenance. TERT has been suggested to act as a chromatin modulator and participate in the transcriptional regulation of gene expression. TERT has also been reported to regulate transcript levels through an RNA-dependent RNA Polymerase (RdRP) activity and produce siRNAs in a Dicer-dependent manner. At the mitochondria, TERT is suggested to protect against oxidative stress-induced mtDNA damage and promote mitochondrial integrity. These extra-telomeric functions of TERT may be advantageous in the context of increased proliferation and metabolic stress often found in rapidly-dividing cancer cells. Understanding the spectrum of non-canonical functions of telomerase may have important implications for the rational design of anti-cancer chemotherapeutic drugs.

scholarly journals Withanolide D Enhances Radiosensitivity of Human Cancer Cells by Inhibiting DNA Damage Non-homologous End Joining Repair Pathway

2020 ◽  
Vol 9 ◽  
Author(s):  
Jerome Lacombe ◽  
Titouan Cretignier ◽  
Laetitia Meli ◽  
E. M. Kithsiri Wijeratne ◽  
Jean-Luc Veuthey ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Human Cancer ◽  
Repair Pathway ◽  
End Joining ◽  
Human Cancer Cells ◽  
Non Homologous End Joining

scholarly journals EXO1 Plays a Role in Generating Type I and Type II Survivors in Budding Yeast

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1641-1649
Author(s):  
Laura Maringele ◽  
David Lydall
Keyword(s):  
Homologous Recombination ◽  
Budding Yeast ◽  
Human Cells ◽  
Telomere Maintenance ◽  
Recombination Process ◽  
Yeast Cells ◽  
Type I ◽  
Type Ii ◽  
Recombination Proteins

Abstract Telomerase-defective budding yeast cells escape senescence by using homologous recombination to amplify telomeric or subtelomeric structures. Similarly, human cells that enter senescence can use homologous recombination for telomere maintenance, when telomerase cannot be activated. Although recombination proteins required to generate telomerase-independent survivors have been intensively studied, little is known about the nucleases that generate the substrates for recombination. Here we demonstrate that the Exo1 exonuclease is an initiator of the recombination process that allows cells to escape senescence and become immortal in the absence of telomerase. We show that EXO1 is important for generating type I survivors in yku70Δ mre11Δ cells and type II survivors in tlc1Δ cells. Moreover, in tlc1Δ cells, EXO1 seems to contribute to the senescence process itself.

Sign in / Sign up

Export Citation Format

Share Document